This application claims priority from Japanese Patent Application Nos. 2000-019080, filed Jan. 27, 2000; and 2000-150578, filed May 22, 2000.
The present invention relates to a D-aminoacylase, a gene encoding the D-aminoacylase, and a method for producing D-amino acids using the D-aminoacylase.
Enzymes have excellent catalytic functions with substrate specificity, reaction specificity, and stereospecificity. Stereospecificity of enzymes, with some exceptions, are nearly absolute.
Recent precise research has increased the importance of optically active substances for use in drugs, pesticides, feeds, and perfumes. Since optical isomers sometimes have quite different biological activities, techniques for specifically obtaining the isomer are important. For example, D(R)-form thalidomide has no teratogenic activity, but its L(S)-form shows strong teratogenicity. The practical use of thalidomide racemate caused the drug injury incidents by thalidomide. Furthermore, if one enantiomer shows an effective biological activity, the other enantiomer may sometimes have no activity, rather, it may competitively inhibit the activity of the effective enantiomer because of the coexistence of both enantiomers. As a result, the biological activity of the racemate is reduced to half or less of the activity of the effective enantiomer. Accordingly, it is industrially important to obtain (synthesize or optically resolve) optically pure enantiomers.
For this objective, an effective procedure has been used widely to optically resolve racemates synthesized. However, an unnecessary enantiomer is always produced as a by-product with the procedure of resolution after synthesis; a problem has been left unsolved in economizing on raw material. Even if the recovered by-product is reused as a raw material, a definite amount of the by-product is always produced. Therefore, enzymatic optical resolution has drawn attention because it does not produce by-products and a bulk of liquid waste. Enzymatic optical resolution is a method of specifically producing a desired enantiomer by utilizing enzyme specificity. Since unnecessary enantiomers are barely synthesized by this method, it is easy to obtain products of high optical purity. In addition, this method is also advantageous in economizing on raw material.
Generally, L-amino acids are widely and largely utilized in seasonings, food and feed additives, and infusions, and are thus very highly demanded. L-amino acids have been produced mainly by direct fermentation using microorganisms. In addition, optical resolution in which N-acyl-DL-amino acids are hydrolyzed with L-aminoacylases is also a known method for producing L-amino acids. It has been utilized to industrially produce L-amino acids that are difficult to produce by fermentation.
L-aminoacylases are widely found in animals, plants, and microorganisms. They have been purified from various organisms, and their properties have been clarified. N-terminal amino acids of many proteins are considered to be N-acetylated in vivo. L-aminoacylases presumably regenerate N-acetyl-amino acids produced by decomposition of proteins to amino acids. Among L-aminoacylases, acylase that acts on N-acyl-L-glutamic acid is reported to be involved in arginine biosynthesis (Fruth et al., J. Gen. Microb, 125:1, 1981).
In contrast, D-amino acids have not been a subject of interest for a long time because they are nonprotein amino acids. D-amino acids were known to naturally occur only in small cyclic peptides, peptidoglycan of bacterial cell walls, and peptide antibiotics. However, D-amino acids have been demonstrated to be constituents of neuro peptides and to exist as binding forms in tooth enamel, the lens, and cerebral proteins, resulting in investigation of physiological significance and enzymatic synthesis of D-amino acids.
At present, DL-amino acids have been optically resolved by physicochemical, chemical, or enzymatic methods. The enzymatic methods are the most convenient and industrially applicable for, for example, continuously producing L-methionine from N-acetyl-DL-methionine using a bioreactor on which L-aminoacylase is immobilized. D-amino acids may also be produced using hydantoinase. The method consists of two-step enzymatic reactions. The first reaction uses D-specific hydantoinase to convert D,L-5-substituted-hydantoin, which is synthesized at low cost from aldehyde analogues, to a D-carbamyl derivative. The second reaction uses D-amino acid carbamylase.
Another method is known in which D-aminoacylase hydrolyzes N-acetyl-DL-amino acids to produce D-amino acids (Sugie et al., Argric. Biol. Chem., 44:1089, 1980; Tsai et al., J. Enzyme Microb. Technol., 14:384, 1992).
D-tryptophan is one of important D-amino acids used as a medicinal raw material and the like. D-tryptophan can be obtained by deacetylating N-acetyl-DL-tryptophan. However, D-aminoacylase capable of efficiently catalyzing this reaction in a stereospecific manner is not yet known.
D-aminoacylase was first reported to be found in Pseudomonas sp. KT83 isolated from soil by Kameda et al. in 1952 (Kameda et al., Nature, 170:888, 1952). This enzyme hydrolyzed N-benzoyl derivatives of D-phenylalanine, D-tyrosine, and D-alanine. Thereafter, D-aminoacylases were derived from microorganisms below.
(Kubo et al., J. Antibiot., 43:550, 1980; Kubo et l., J. Antibiot. 43:556, 1980; Kameda et al., Chem. Pharm. Bull., 26:2698, 1978; Kubo et al., J. Antibiot. 43:543, 1980)
(Sugie et al., Argric. Biol. Chem., 42:107, 1978; Sugie et al., Argric. Biol. Chem., 44:1089, 1980)
(Tsai et al., Appl. Environ. Microbiol., 54:984, 1988; Yang et al., Biosci. Biotech. Biochem., 56:392, 1992; Yang et al., Appl. Environ. Microbiol., 57:2767, 1991; Tsai et al., Microb. Technol., 14:384, 1992; Moriguchi et al., Appl. Environ. Microbiol., 54:2767, 1988; Sakai et al., FEBS, 289:44, 1991; Sakai et al., J. Ferment. Bioeng., 71:79, 1991; Sakai et al., Appl. Environ. Microbiol., 57:2540, 1991; Moriguchi et al., Biosci. Biotech. Biochem., 57:1145, 1993; Kayama et al., J. Biochem., 118:204, 1995; Moriguchi et al., Biosci. Biotech. Biochem., 57:1149, 1993)
Tsai et al. and Moriguchi et al. also clarified the characteristics of D-aminoacylase derived from microorganisms belonging to the genera Alcaligenes and Pseudomonas and the amino acid and nucleotide sequences of the enzymes. Moriguchi et al. found, by using different inducers, three types of D-aminoacylases derived from microorganisms belonging to the genera Alcaligenes and Pseudomonas (Wakayama et al., Biosci. Biotech. Biochem., 59:2115, 1995).
Furthermore, Moriguchi et al. determined the nucleotide sequences of these D-aminoacylases derived from a microorganism belonging to the genus Alcaligenes and compared them with L-aminoacylases derived from Bacillus stereothermophilus, human, and pig. The result demonstrated that these D-aminoacylases have a low homology with L-aminoacylases (Wakayama et al., Biosci. Biotech. Biochem., 59:2115, 1995).
As to Actinomycetes, Sugie et al. reported D-aminoacylase of a microorganism belonging to the genus Streptomyces (Sugie et al., Argric. Biol. Chem., 44:1089, 1980). However, the enzyme has not been purified yet, and its characteristics remain unknown.
Any of these known D-aminoacylases exhibit only low activities for N-acetyl-D-tryptophan and cannot be used for synthesizing D-tryptophan. With these points described above as background, it has been demanded to isolate D-aminoacylase capable of producing D-tryptophan stereospecifically using N-acetyl-DL-tryptophan as a substrate as well as a gene encoding the enzyme.
An objective of the present invention is to provide D-aminoacylase capable of producing D-tryptophan stereospecifically using N-acetyl-DL-tryptophan as a substrate and a gene encoding the enzyme. Another objective of the present invention is to provide uses of the D-aminoacylase and the gene.
In order to achieve the objectives mentioned above, the present inventors have repeatedly isolated many D-aminoacylases from a wide variety of biological species and investigated substrate-specificities of the enzymes. As a result, the inventors have found that a fungus belonging to the genus Hypomyces has the activity to produce D-amino acid from N-acetyl-D-amino acid, namely, D-aminoacylase activity. Subsequently, the present inventors have succeeded in separating and purifying D-aminoacylase from the fungus with D-aminoacylase activity by using ammonium sulfate precipitation and various chromatographic methods. In addition, the present inventors elucidated various physicochemical properties such as substrate specificity and thermal stability of the purified D-aminoacylase and have found that it is possible to produce D-amino acids efficiently by incubating the fungus-derived D-aminoacylase with N-acetyl-D-amino acids under proper conditions. It has been confirmed that the D-aminoacylase discovered by the present inventors has especially high enzymatic activity for N-acetyl-D-tryptophan and that the enzyme excels in industrial applicability.
Furthermore, the present inventors have isolated this D-aminoacylase and a gene encoding the enzyme, and revealed their structure. Thus, the present inventors completed the present invention. Previously identified D-aminoacylases have only low enzymatic activities for N-acetyl-D-tryptophan and are thus useless for D-tryptophan synthesis.
Specifically, the present invention relates to a polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 1;
(b) a polynucleotide encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2;
(c) a polynucleotide hybridizing to a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 under a stringent condition, wherein said polynucleotide encodes a polypeptide having activity of a D-aminoacylase having physicochemical properties of (i) and (ii) below; and
(d) a polynucleotide encoding a polyopeptide having the amino acid sequence set forth in SEQ ID NO: 2 in which one or more amino acid are substituted, deleted, inserted, and/or added, wherein said polynucleotide encodes a polypeptide having activity of a D-aminoacylase having physicochemical properties of (i) and (ii) below
(i) action: the enzyme acts on N-acetyl-D-amino acids to produce the corresponding D-amino acids and
(ii) substrate specificity: the enzyme acts on N-acetyl-D-tryptophan, N-acetyl-D-phenylalanine, N-acetyl-D-valine, N-acetyl-D-leucine, and N-acetyl-D-methionine, but not on N-acetyl-L-tryptophan, N-acetyl-L-phenylalanine, N-acetyl-L-valine, N-acetyl-L-leucine, or N-acetyl-L-methionine.
The present invention also relates to a polypeptide encoded by the above polynucleotide.
Furthermore, the present invention features a vector comprising the above polynucleotide, a transformant expressively carrying the polynucleotide, or the vector, and a method of producing the polypeptide, which comprises culturing the transformant and recovering the expression product. The transformant is preferably derived from E. coli. 
Another feature of the present invention is a polynucleotide hybridizing to the polynucleotide set forth in SEQ ID NO: 1 or the complementary strand thereof, wherein said polypeptide comprises at least 15 nucleotides. This polynucleotide can be used as a primer for synthesizing the above-mentioned polynucleotide or used as a probe for detecting the polynucleotide.
Still another feature of the present invention is a method for synthesizing D-amino acids, which comprises contacting the above-mentioned polypeptide or the transformant with N-acyl-DL-amino acid represented by the formula (I) or its salt: 
wherein R1 and R2 may be identical or different and each represents a hydrogen atom or a substituted or unsubstituted hydrocarbon group; provided that R2 does not represent a hydrogen atom; and X is H, NH4, or a metal ion. In the formula (I), R1 and R2 preferably each represents an alkyl, alkenyl, alkynyl, cycloalkyl, aryl, or aralkyl group, or the derivative thereof. More preferably, R1 is an xcex2-methylindolyl, benzyl, thiomethylethyl, isopropyl, or 2-methylpropyl group and R2 is a methyl, chloromethyl, phenyl, or aminomethyl group.
xe2x80x9cD-aminoacylasexe2x80x9d used herein means an enzyme reacting with an N-acyl-D-amino acid to catalyze the production of an organic acid and a D-amino acid.